The ability to grow and study various biological materials, such as mammalian cells, is important in the laboratory and industry. However, many cells and other biological materials will grow only if they can attach to an appropriate substrate (i.e., the biological material exhibits an adherent phenotype). The ability of many biological materials to attach and grow on a substrate is dependent upon the substrate being chemically activated to be somewhat hydrophilic. Hydrophilicity may be provided by various functional groups on the substrate. These functional groups include hydroxyl (OH), carboxyl (COOH), and amine (NH2 and NH) groups, for example.
Preparing such a chemically activated substrate generally involves (1) the application of energy in the form of discharge of either corona or plasma to the substrate, or (2) the application of a coating (such as poly-D-lysine or collagen) to the substrate. These processes provide groups to which biological materials may attach. For example, in corona discharge, the substrate being treated is exposed to an electrical discharge, or corona. Oxygen molecules within the discharge area break into their atomic form and are free to bond to the ends of molecules of the substrate being treated, resulting in a desired chemically activated substrate. The main advantage of corona or plasma discharge treatment of substrates is ease of manufacture; polymeric substrates subjected to such energetic discharge may be easily treated in-line and sterilized.
However, both corona and plasma discharge treatments have drawbacks. For example, corona discharge is limited to bonding of oxygen within the first few nanometers of the substrate, and can be wiped off. Thus, the substrate can be degraded rapidly. Additionally, corona discharge treatment can generally only provide only up to about 20%, at best, surface oxygen on the substrate. Certain biological materials, such as certain cell lines, require more oxygen. While plasma discharge can produce higher oxygen levels than corona discharge, it requires the substrate to be treated in a vacuum.
Treating substrates by the application of various biologic coatings, such as poly-D-lysine or collagen, mimic the milieu on which cells would typically grow. However, these coatings often do not survive sterilization by gamma irradiation. Additionally, the biological origin of these coatings raises the possibility of disease transmission.
Energetic discharge and coating treatments can provide only a few standard activated substrates for use with all biological materials, producing little variation in the surface chemistry of substrates used to culture biological materials. This is problematic because, to some degree, every cell line or other biological material exhibits a preference for a certain type, mixture, and/or ratio of functional groups on a culture substrate. For example, some exhibit a preference for multiple different functional groups, while some exhibit a preference for only one functional group in a specific concentration range. However, as described above, current technologies generally employ “one-size-fits-all” strategies (e.g., a substrate with corona treatment used for all types of cell lines). The resulting substrates can, at best, be controlled to produce a range of surface chemistry, rather than being able to produce any chemistry. For example, corona treatment, even at low power, has a characteristic surface chemistry that is somewhat dependent upon atmospheric conditions at the time (humidity, temperature, and volatile organics in the air). Ultimately, it generally produces a substrate having a range of oxygen concentrations from between 10%-20%. However, it cannot produce substrates having high oxygen concentrations, nor can it produce substrates including other or additional functional groups, such as amine groups. Such a substrate may not be adequate for a particular biological material that exhibits a preference for high oxygen levels and/or amine groups.
Further, many biological materials are grown in culture apparatus that not only have substrates treated as described above, but may also include serum to enhance growth. For example, the human embryonic kidney (HEK) 293 cell line is increasingly used because it is easy to grow, is genetically stable, has a normal complement of human chromosomes, and incorporates portions of the adenovirus genome that make it amenable for genetic manipulation. Other cell lines exhibiting similar characteristics are PER C6, Vero, BHK-21, and MDCK.
HEK cells can be successfully grown under conditions recommended by the American Type Culture Collection (ATCC) (e.g., in Dulbecco's Modified Eagle's Media (DMEM)+10% fetal bovine serum (FBS)) on standard cell culture substrates (e.g., those subjected to corona treatment). Serum enhances HEK cell growth and as such, it has been included in most cell culture procedures. Recently, however, safety concerns have been raised and it is the single most expensive component of most media formulations. While it is desirable to reduce or eliminate the need for serum altogether, the ability of almost all cell lines, including HEK cells, to grow on corona treated substrates diminishes as the serum concentration is reduced.
Recently, the Corning product CellBIND®, is being offered in all standard cell culture formats. CellBIND® includes a substrate described as supporting growth of adherent cells without serum. It has been suggested that the enhanced ability of CellBIND® to grow HEK cells is primarily a result of its higher quantity of oxygen, specifically hydroxyl groups, relative to corona treated substrates (15% oxygen on CellBIND® vs. 8% oxygen on corona treated substrates) and to a lesser but still significant degree by the amount of carboxylic acids (7% carboxylic acid in CellBIND® vs. 2% carboxylic acid on corona treated substrates). However, CellBIND® does not include other functional groups such as amine groups, and it is not tailored to different preferences that may be exhibited by different biological materials.
Other products and methods are desirable. For example, an apparatus and method to control the chemistry of an adherent substrate without secondary treatment (e.g., corona discharge) and/or to improve the quality of existing secondary treatments would be desirable. Further, an apparatus including functional groups on a polymeric substrate that is not subject to degradation and can be used with reduced serum or without serum would be desirable. Further, a method of preparing an apparatus having a substrate that varies in functional groups, both in type and concentration, in order to tailor the apparatus to a particular biological material would be desirable.